The epithelial Na channel (ENaC) is a key mediator of the renal control of salt balance regulation. We will explore the mechanisms involved in this process. The proposed work has three related major aims. Aim 1. Trafficking of ENaC in the kidney. We have shown directly that salt depletion and aldosterone administration lead to increased surface expression of ENaC subunits. This comes about at least in part from increased rates of delivery of channels to the plasma membrane. We will address several questions: A. Is the ER a point at which ENaC is either degraded or moved forward to the Golgi apparatus? We will examine ubiquitination of ENaC as well as its in a mouse model of Liddle's syndrome. B. Is ENaC maturation in the Golgi accelerated by aldosterone? We will examine ENaC expression and processing in the cis- and trans-Golgi with acute and chronic elevation of aldosterone. C. Do endosomes control surface expression? We will assess ENaC in early endosomes and exosomes under conditions of acute salt repletion and ADH stimulation. D. Does ENaC in urinary exosomes represent excretion of channel protein from multi-vesicular bodies? We will examine whether MVB markers associate with ENaC in exosomes. Aim 2. Role of trafficking in the response to aldosterone. We have shown that increased ENaC surface expression is an early event in the response to aldosterone, and that later increases in ENaC function may reflect activation of channels at the surface. To follow up these findings we will: A. assess the complete time course of changes in whole-cell currents, single-channel currents, surface expression and exosome excretion of ENaC. B. confirm the rapid trafficking of ENaC in response to acute administration of diuretics C. test whether changes in ENaC function and trafficking contribute to diurnal variations in Na excretion. Aim 3. Role of aldosterone-induced proteins in the regulation of ENaC. Most effects of aldosterone on the kidney depend on changes in gene expression, including SGK1 and GILZ. We have shown that induction the serine-/threonine kinase SGK1 is essential for trafficking of ENaC in response to aldosterone but that ENaC activity can be increased in the absence of this protein. We will: A. test the idea that SGK1 is required for early but not late effects of aldosterone. B. examine the role of a second induced protein, GILZ, in aldosterone-dependent ENaC regulation C. use RNA sequencing to distinguish early and late aldosterone-dependent genes.